Preparation of pellets containing fungi for control of soilborne diseases

ABSTRACT

This invention relates to a method for preparing pellets containing living biocontrol fungi. Fungi are selected and grown for sufficient time to produce inoculum. The fungal propagules are harvested, homogenized and diluted with sodium alginate solution. Pelletization is then accomplished by dropwise addition of the fungal propagule-alginate mixture into a solution of calcium chloride or calcium gluconate. The resultant alginate gel pellets containing living fungi can then be dried and are used to inoculate agricultural fields infested with soilborne plant diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 506,952, filedJune 22, 1983 now abandoned.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to formulations of inoculum of microorganisms.

(2) Description of the Prior Art

Antagonistic fungi in several genera have been previously shown toeffectively control plant disease caused by pathogenic fungi.Talaromyces flavus is used to control Verticillium wilt of eggplantwhich is caused by Verticillium dahliae (J. J. Marois, et al., 1982.Plant Disease 66:1164-1168). Since eggplant is usually subjected totransplanting, T. flavus is applied as a drench before transplantingtakes place.

Potatoes and cotton are other exmaples of economically important cropswhich are also subject to V. dahliae. Since potatoes and cotton are bothseeded directly into the field, the drench formulations are notpracitcal. However, dust formulations of T. flavus have been used totreat potato seed pieces but this is inefficient because the dust easilyblows away. In addition, the dust is not adequately distributed in theroot zone where it is most needed to protect the roots from thepathogen.

Connick, Jr., (U.S. Pat. Nos. 4,401,456 and 4,400,391) both discloseprocesses for incorporating chemical, non-living, bioactive materials inalginate gels. Connick, Jr., (U.S. Pat. Nos. 4,401,456 and 4,400,391)discloses chemicals which are formulated to kill living matter. He alsoteaches the use of cations in the process, i.e. barium, copper, lead,zinc, all of which would be reasonably expected to kill any living fungiincorporated into the alginate gel.

The use of alginate gel technology to formulate agricultural products,pesticides and food items has also been disclosed. For example, U.S.Pat. No. 4,053,627 describes the use of alginate gel discs for mosquitocontrol, U.S. Pat. No. 3,649,239 discloses fertilizer compositions, andU.S. Pat. No. 2,441,729 teaches the use of alginate gels as insecticidalas well as candy jellies. None of these patents disclose any method forincorporating living materials or fungi into an alginate gel matrix.

SUMMARY OF THE INVENTION

Pelletization of living biocontrol fungi useful to control selectedplant disease using aqueous solutions of sodium alginate, calciumgluconate and calcium chloride is disclosed. The granular formulationsof living fungi produced by this method have extended shelf life andsustained release or sustained production characteristics.

The method comprises the following steps in combination: selecting andgrowing fungi for sufficient time to be used as inoculum, harvesting andhomogenizing the fungal propagules for sufficient time to uniformly mixthe propagules, and diluting the fungus propagule homogenate with asodium alginate solution with a sufficient concentration of sodiumalginate to effect adequate gelation. This mixture is then addeddropwise to a solution of CaCl₂ or calcium gluconate, thus formingalginate gel pellets which contain fungus dispersed throughout.

U.S. Pat. Nos. 4,401,456 and 4,400,391 teach a process for incorporatingchemical bioactive materials in alginate gels. There is no teaching insaid patents on, nor is there anticipated, the incorporation of livingfungi as active materials. Indeed, one skilled in the art might expectfungi to die or become ineffective as a result of being incorporated inalginate pellets. The chemical bioactive materials of said U.S. Pat.Nos. 4,401,456 and 4,400,391 are released from the products by virtue oftheir water solubility (leaching or diffusion) or as a result ofbiodegradation of the alginate matrix. This is totally different fromthe growth and release of active propagules from a living fungus.

It was completely unexpected that a living fungus could be incorporatedin alginate gel pellets or granules to give an effective material. Forexample, barium chloride and cupric chloride are among the preferredwater-soluble metal salt gellants taught by U.S. Pat. Nos. 4,401,456 and4,400,391 for use with chemical bioactive materials, but these salts arefungitoxic when used in the process of the present invention. Otherreasons why it is not obvious to use the teachings of U.S. Pat. Nos.4,401,456 and 4,400,391 to produce effective fungus-containing pelletsare stated below.

All mechanisms known for biocontrol by fungi (competition, antibiosisand parasitism) require an actively metabolizing antagonist. Hence, theeffectiveness of biocontrol fungi pelletized in an alginate matrix couldnot be expected since propagules of the fungi must grow free of thematrix to control pathogenic fungi.

The ability of the biocontrol fungi to survive the process of thepresent invention could not be predicted a priori because one would haveexpected the osmotic shock resulting from addition of the liquidalginate suspension into the calcium salt to kill the biocontrol fungi.

Stability of alginate gel pellets containing living fungi is veryimportant for commerical reasons. The biocontrol fungi must survive forextended shelf life periods of time to meet shipping needs andagricultural uses. Furthermore, other contaminant microorganisms arealso pelletized along with the desired fungi during the formulationprocess. Since many microorganisms may be capable of competing with thedesired fungi, the recovery and growth of the desired fungi afterstorage was impossible to predict beforehand. Applicants' biocontrolfungi-containing pellets produced living colonies of fungi when appliedto an agricultural enviornment after extensive shelf life periods oftime. This was totally unpredictable.

In addition to its use in control of plant pathogens, the method ofsodium alginate formulation described by applicants' preferredembodiment has other applications. For example, the production ofinoculum of plant pathogens would facilitate uniform infestation of anarea with a pathogen to evaluate host resistance, control methods, or toperform epidemiological research.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Soilborne diseases of agricultural crops can be controlled by thefollowing biocontrol fungi:

Gliocladium virens isolate G1-3 (NRRL #15937)

Penicillium oxalicum isolate Windels (NRRL #15938) (ATCC #52658)

Talaromyces flavus isolate Tf-l (NRRL #15935) (ATCC #52201)

Talaromyces flavus biotype Tfl-1 (NRRL #15936)

Trichoderma viride biotype T-1-R9 (ATCC #52442) (U.S. Pat. No.4,489,161).

All, except the T. viride, are on deposit with the Agricultural ResearchCulture Collection (NRRL) and available from: A. J. Lyons, Curator, ARSPatent Collection, Culture Collection Research, NRRC, 1815 N. UniversityStreet, Peoria, Ill., 61605. In addition, the P. oxalicum, T. flavusTf-1, and the Trichoderma viride are also on deposit with: American TypeCulture Collection, Rockville. Md. In addition, the T. viride T-l-R9 isa genetically manipulated biotype and has been patented (U.S. Pat. No.4,489,161).

Each of the five fungi are cultured and pelletized separately todemonstrate the flexibility of the process. Since two of the fungiproduced two distinct types of spores, they are also pelletizedseparately to demonstrate further flexibility. The versatility of theprocess is also demonstrated by using two different biotypes of the samefungus.

Ascospores are produced by growing T. flavus on potato-dextrose agar for4 weeks at 30° C. in the dark. When T. flavus is grown on amolasses-corn steep medium (a more carbon rich medium) in the light at25° C. for 1 week, conidia are produced. Conidia of G. virens areproduced by growing the fungus on V-8 juice agar for 1 week at 25° C.Conidia of P. oxalicum and T. viride are produced by growing theorganisms on potato-dextrose agar for 1 week at 25° C. Othermicrobiological media are also suitable and can even be incorporatedinto the pellets along with the propagules. Experimental details forgrowing the fungi are given in the article "Biological Control ofVerticillium wilt of eggplant in the field," by J. J. Marois, et al.,Plant Disease. 66:1166-1168 (1982).

Propagules of the fungi are dislodged from the agar surface by rubbing,suspended in water, then comminuted in a Sorval mixer or similarapparatus to insure uniform distribution of the propagules. The alginatesolution which contains 10 g of sodium alginate, 100 g of pyrophyllite,and 1 L of water is mixed for one minute in a blender and then variousconcentrations of propagules in water are added. The resulting mixtureis then added dropwise to a salt solution of either 0.25 M CaCl₂ or 0.1M C₁₂ H₂₂ CaO₁₄ (calcium gluconate) in distilled water. Pellets formalmost immediately upon contact with the salt solution. The saltsolution is decanted and the pellets are dried. Drying to a 5% to 15%moisture content is preferred. Pellets can be disintegrated in mixtureof 8.7×10⁻³ M. KH₂ PO₄ and 3.0×10⁻² M. Na₂ HPO₄ (pH 7.7) and assayed bydilution plating to determine viable propagule population. Sodiumalginate is the preferred alginate but other water soluble salts ofalginic acid such as potassium alginate may be used. Sodium alginateconcentration in the propagule-sodium alginate-pyrophyllite-watermixture can be from 0.5 to 2.0% (w/v) but the preferred concentration is0.75 to 1.0%.

A water soluble calcium salt such as calcium chloride or calciumgluconate is necessary for gelation of sodium alginate solutions, andthese compounds are not toxic to the fungi. An effective concentrationrange of the calcium chloride or calcium gluconate bath, also called thesalt or gellant solution, is 1% to 15% (w/v), but 2% to 5% is preferred.Gelation proceeds faster as the concentration of the salt solution isincreased.

Various organic and inorganic fillers such as clays, diatomaceous earth,sand, or corn cobs can be used in the formulations of the presentinvention. Pyrophyllite (hydrous aluminum silicate) is preferred inquantities up to about 20% (w/v) because it has a neutral pH. Otheradjuvants that may be of use when incorporated in formulations are:selective fungistats, antibiotics, nutrients, materials that stimulatespore production, viscosity modifiers, and materials to control hardnessof the pellets or their rate of biodegradation or disintegration.

The simplicity of the requirements for carrying out the process of thepresent invention permits much latitude in equipment design. A suitableapparatus, described only for the purpose of illustration and not to beconstrued as limiting to the invention, consists of a reservoir tocontain the alginate-propagule-prophyllite mixture, a pump to feed thismixture, or a gravity-feed arrangement, from the reservoir to orificesabout 1-2 mm in diameter that permit the mixture to be added in adropwise manner into a gellant solution contained in any convenientvessel. The alginate gel pellets that form have propagules of thedesired fungus incorporated throughout and are harvested from thegellant solution by any suitable means. The alginate gel pellets may bedried to form dried pellets or granules making them more suitable forstorage and agricultural field use. A continuous process is possibleinvolving the continuous removal of gel pellets and maintenance of aneffective gellant solution concentration. It is also possible to extrudethe alginate-propagule-pyrophyllite mixture into the gellant solution toform a string-like gel which could be further processed to makegranules.

Most desired fungi should be processed below 50° C., preferably in therange of 15-40 C. Dwell time of the gel pellets in the gellant solutioncan be from about 0.1 to 60 minutes, but 0.2 to 5 minutes is preferred.

The fungi reproduced when the gel pellets were placed in moist fieldsoil or on appropriate microbiological media when brought into contactwith moisture. Dried pellets may be stored for an extended period oftime. (Table 1).

Pellets produced as described above have sustained-releasecharacteristics and provide residual activity to enhance the performanceof the biocontrol fungi. Applicants' preferred embodiment also providesa useful method for storing inoculum for extended periods of time.

The pathogens enumerated above, when formulated as dried pellets,reproduced readily under field conditions when adequate moisture waspresent and effectively controlled the targeted soilborne diseases.

Alginate formulations of biocontrol fungi may be applied directly tosoil as pellets, or propagules can be produced on the pellets, thenremoved and applied to the desired environment using any compatible,effective means of distribution.

Formulation and production of alginate gel pellets containing theabove-described living biocontrol fungi are readily illustrated in thefollowing examples:

EXAMPLE 1

Ascospores of Talaromyces flavus isolate Tf-1 were produced by growingthe fungus on potato-dextrose agar for 4 weeks in the dark at 30° C.Cleistothecia were removed by gently scraping the medium and were addedto water and comminuted for 1 minute in a Sorvall mixer to insuredisruption of cleistothecia and asci and produce a uniform distributionof ascospores. Ascospores were added to a mixture containing 1% byweight of sodium alginate and 10% of pyrophyllite in sufficientdistilled water to total 200 ml in volume. The ascospores were added inconcentrations ranging from 10⁵ to 10⁸ per ml of alginate suspension.The alginate-pyrophyllite-fungal propagule mixture was stirred and addeddropwise from a reservoir through tubes (each terminating in a 1 mmdiameter orifice), the droplets falling a distance of about 15 cm into200 ml of a 0.25 M CaCl₂ gellant solution. Gel beads immediately formed,allowed to remain in the gellant solution for up to 45 minutes,separated by decanting, spread on aluminum foil, and dried to about 10%moisture content at ambient temperature using a gentle flow of air.About 22 g of pellets were obtained. Drying transformed the gel beadsinto hard granules or pellets which were stored under room conditions.Viable propagule populations were determined as follows: beforepelletization, immediately following pelletization, 24 hours, 1,2,4,8,and 12 weeks. Results reported in Table 1 show that 100% of the funguspropagules survived during pellet formation.

EXAMPLE 2

Conidia of Talaromyces flavus isolate Tf-1 were produced by growing thefungus on molasses-corn-steep medium in the light at 25° C. for 1 week.The propagules were pelletized and viable populations of funguspropagules were determined as in Example 1 with results as reported inTable 1 which shows 100% survival during pellet formation.

EXAMPLE 3

Ascospores of Talaromyces flavus isolate Tf-l were grown and pelletizedas described in Example 1 except that 0.1 M calcium gluconate was usedas the gellant solution and only one initial concentration of spores(10⁶ per M alginate suspension) was pelletized. Viable populations offungus propagules were determined as in Example 1 with results reportedin Table 1 which shows 100% survival during pellet formation.

EXAMPLE 4

Conidia of Talaromyces flavus isolate Tf-l were produced by growing thefungus on molasses corn-steep-medium in the Tight at 25° C. for 1 week.The propagules were pelletized as described in Example 1 except that 0.1M calcium gluconate was used as the gellant solution. Only one initialconcentration (10⁶ per ml adequate suspension) was pelletized. Viablepopulations of fungus propagules were determined as in Example 1 withresults reported in Table 1 which shows 100% survival during pelletformation.

EXAMPLE 5

Ascospores of Talaromyces flavus isolate Tfl-1 were grown and pelletizedas in Example 1. Viable populations of fungus propagules were determinedas in Example 1 with results reported in Table 1 which shows 100%survival during pellet formation.

EXAMPLE 6

Conidia of Talaromyces flavus isolate Tfl-1 were grown and pelletized asin Example 2. Viable populations of fungus propagules were determined asin Example 1 with results reported in Table 1 which shows 100% survivalduring pellet formation.

EXAMPLE 7

Ascospores of Talaromyces flavus isolate Tfl-1 were grown and pelletizedas in Example 3. Viable populations of fungus propagules were determinedas in Example 1 with results reported in Table 1 which shows 100%survival during pellet formation.

EXAMPLE 8

Conidia of Talaromyces flavus isolate Tfl-1 were grown and pelletized asin Example 4. Viable populations of fungus propagules were determined asin Example 1 with results reported in Table 1 which shows 100% survivalduring pellet formation.

EXAMPLE 9

Conidia of Gliocladium virens isolate Gl-3 were produced by growing thefungus on V-8 juice agar for 1 week at 25° C. Light was not excluded.Conidia were pelletized as described in Example 1. Viable populations offungus propagules were determined as in Example 1 with results reportedin Table 1 which shows 40-89% survival during pellet formation.

EXAMPLE 10

Conidia of Gliocladium virens isolate Gl-3 were grown on potato-dextroseagar and pelletized as described in Example 1 except that 0.1 M calciumgluconate was used as the gellant solution. Viable populations of funguspropagules were determined as in Example 1 with results reported inTable 1 which shows a 57% survival during pellet formation.

EXAMPLE 11

Conidia of Penicillium oxalicum were grown on potato-dextrose agar andpelletized as described in Example 1. Viable populations of funguspropagules were determined as in Example 1 with results reported inTable 1 which shows 100% survival during pellet formation.

EXAMPLE 12

Conidia of Penicillium oxalicum were grown on potato dextrose agar andpelletized as described in Example 1 except that 0.1 M calcium gluconatewas used as a gellant solution. Viable populations of fungus propaguleswere determined as in Example 1 with results reported in Table 1 whichshows 100% survival during pellet formation.

EXAMPLE 13

Conidia of Trichoderma viride isolate T-1-R9 were grown onpotato-dextrose agar and pelletized as described in Example 1. Viablepopulations of fungus propagules were determined as in Example 1 withresults reported in Table 1 which shows 100% survival during pelletformation.

EXAMPLE 14

Conidia of Trichoderma viride isolate T-l-R9 were grown on potatodextrose agar and pelletized as described in Example 1 except that 0.1 Mcalcium gluconate was used as the gellant solution. Viable populationsof fungus propagules were determined as in Example 1 with resultsreported in Table 1 which shows 100% survival during pellet formation.

The following examples demonstrate effective agricultural use of theabove-described biocontrol fungi:

EXAMPLE 15

Pellets of Talaromyces flavus isolate Tf-l, as prepared in Example 1,were applied at a rate of 6.7 g pellets/100 row feet, in an agriculturalfield naturally infested with Verticillium dahliae. These furrows werethen planted with potatoes. Incidence of Verticillium wilt was recorded2 weeks prior to harvest. There was approximately 60% less wilt and 15%increase in the weight of marketable tubers in the T. flavus-pellettreated potatoes compared to nontreated replicates.

EXAMPLE 16

Pellets containing Gliocladium virens as prepared in Example 9 wereincorporated into field soil naturally infested with Pythium andRhizoctonia (causes pre- and post- emergence damping off disease).Pellets were incorporated at the rate of 0.5% (w/w). Radish seeds wereplanted and after 10 days there were nearly twice as many plants in theGliocladium treated soil as compared to a control.

EXAMPLE 17

Pellets containing Gliocladium virens as prepared in Example 10 weretested for control of damping-off as described in Example 16. More thantwice as many healthy seedlings survived using the pellets containingthe biocontrol fungus than in the replicates receiving pellets madewithout fungus.

EXAMPLE 18

Pellets containing Penicillium oxalicum as prepared in Example 11 weretested for control of damping-off as described in Example 16. There werealmost twice as many healthy seedlings in the P. oxalicum-pellet treatedreplicates as in the control treatments.

EXAMPLE 19

Pellets containing Penicillium oxalicum as prepared in Example 12 weretested for control of damping-off as described in Example 16. There weremore than twice as many healthy seedlings in the P. oxalicum-pellettreatments as in those treated with pellets containing no fungalpropagules.

EXAMPLE 20

Pellets containing Trichoderma viride as prepared in Example 13 weretested for control of damping-off as described in Example 16. There weretwice as many healthy seedlings in the T. viride-pellet treatments as inthose treated with pellets containing no fungal propagules.

EXAMPLE 21

Pellets containing Trichoderma viride as prepared in Example 14 weretested for control of damping-off as described in Example 16. There weremore than twice as many healthy seedling in the T. viride-pellettreatments as in those treated with pellets containing no fungalpropagules.

                                      TABLE 1                                     __________________________________________________________________________    SURVIVAL OF FUNGI USED AS BIOLOGICAL                                          CONTROL AGENTS IN ALGINATE PELLETS AT ROOM TEMPERATURE                                        Concentration                                                                           Survival                                                            Before Pellet                                                                           During                                                              Formation Pellet       Propagule Viability After                              (Equivalent                                                                             Formation                                                                             ES.sub.50.sup.a                                                                    12 Weeks                               Isolate and Propagule                                                                    Gellant                                                                            Propagules/Pellet)                                                                      (% Per Pellet)                                                                        (Weeks)                                                                            Per g × 10.sup.3                                                               Per Pellet                      __________________________________________________________________________    Gliocladium virens                                                                       .sup. CC.sup.b                                                                     .sup. 6.7 × 10.sup.5c                                                             89.0    0.2  0.18   12                              (Gl-3) Conidia  2.0 × 10.sup.5                                                                    40.0    0    0.45   30                                              3.4 × 10.sup.4                                                                    78.4    0.2  1.10   73                                         CG   3.4 × 10.sup.4                                                                    57.0    2.2  35.00*.sup.d                                                                         2.3 × 10.sup.3            Penicillum oxalicum                                                                      CC   4.3 × 10.sup.6                                                                    100.0   0    0.08   5                               Conidia         4.4 × 10.sup.5                                                                    100.0   0    0.12   8                                               9.3 × 10.sup.3                                                                    100.0   1.9  0.05   3                                          CG   9.3 × 10.sup.3                                                                    100.0   >12.0                                                                              8.50*  5.7 × 10.sup.2            Talaromyces flavus                                                                       CC   2.7 × 10.sup.4                                                                    100.0   >12.0                                                                              220.00 1.5 × 10.sup.4            (Tfl) Ascospores                                                                              3.3 × 10.sup.3                                                                    100.0   >12.0                                                                              17.50  1.2 × 10.sup.3                            1.7 × 10.sup.2                                                                    100.0   6.4  5.50   3.7 × 10.sup.2                       CG   1.7 × 10.sup.2                                                                    100.0   >12.0                                                                              390.00*                                                                              2.6 × 10.sup.4            Conidia    CC   1.5 × 10.sup.6                                                                    100.0   0    0.12   8                                               1.3 × 10.sup.4                                                                    100.0   5.4  0      0                                               1.5 × 10.sup.3                                                                    100.0   0    0      0                                          CG   1.5 × 10.sup.3                                                                    100.0   8.6  405.00*                                                                              2.7 × 10.sup.4            Talaromyces flavus                                                                       CC   2.7 × 10.sup.4                                                                    100.0   0.7  0      0                               (Tfl-1) Ascospores                                                                            2.0 × 10.sup.3                                                                    100.0   4.0  0      0                                               3.9 × 10.sup.2                                                                    100.0   5.5  2.00   1.3 × 10.sup.2                       CG   3.9 × 10.sup.2                                                                    100.0   >12.0                                                                              255.00*                                                                              1.7 × 10.sup.4            Talaroymces flavus                                                                       CC   2.0 × 10.sup.7                                                                    100.0   0    0.10   7                               (Tfl-1) Conidia 2.6 × 10.sup.5                                                                    100.0   9.7  0      0                                               7.3 × 10.sup.3                                                                    100.0   0    0.20   13                                         CG   7.3 ×  10.sup.3                                                                   100.0   2.8  255.00*                                                                              1.7 × 10.sup.4            Trichoderma viride                                                                       CC   6.8 × 10.sup.6                                                                    100.0   2.7  0      0                               (T-1-R9) Conidia                                                                              6.7 × 10.sup.5                                                                    100.0   2.7  0      0                                               6.6 × 10.sup.4                                                                    100.0   2.5  0      0                                          GC   6.6 × 10.sup.4                                                                    100.0   >12.0                                                                              940.00*                                                                              6.4 × 10.sup.4            __________________________________________________________________________     .sup.a ES.sub.50 = Effective Survival 50% indicates the length of time        after drying for loss of 50% of the viable propagules.                        .sup.b CC = calcium chloride; CG = calcium gluconate.                         .sup.c Data from one experiment.                                              .sup.d Values marked by an asterisk are significantly greater (P ≦     0.05) compared to population values for their respective initial, airdry      concentrations.                                                          

We claim:
 1. Alginate gel pellets containing living fungi inoculumdispersed throughout said fungi targeted to control selected soilborneplant diseases; said pellets comprising effective concentrations ofliving fungi which are selected to effectively control targetedsoilborne plant diseases as the active ingredient incorporated into analginate gel matrix carrier.
 2. The alginate gel pellets of claim 1wherein the living fungi is selected from the group consisting of:Gliocladium virens isolate Gl-3, Penicillium oxalicum isolate Windels,Talaromyces flavus isolate Tf-1, Talaromyces flavus biotype Tfl-l andTrichoderma viride biotype T-l-R9.
 3. The alginate gel pellets of claim2 including a pyrophyllite filler material.
 4. Dried and hardenedalginate gel pellets of claim
 3. 5. The alginate gel pellets of claim 4wherein the living fungus is Gliocladium virens isolate Gl-3 selected tocontrol damping-off diseases.
 6. The alginate gel pellets of claim 4wherein the living fungus is Penicillium oxalicum isolate Windelsselected to control damping-off diseases.
 7. The alginate gel pellets ofclaim 4 wherein the living fungus is Talaromyces flavus isolate Tf-lselected to control Verticillium wilt disease.
 8. The alginate gelpellets of claim 4 wherein the living fungus is Talaromyces flavusbiotype Tfl-1 selected to control Verticillium wilt disease.
 9. Thealginate gel pellets of claim 4 wherein the living fungus is Trichodermaviride biotype T-1-R9 selected to control damping-off diseases.